A nanoscale interface able to induce molecular and functional differentiation of cultured rat primary astrocytes in vitro

Astroglial ion channels and aquaporins are key player in the maintenance of cerebral homeostasis. Alteration in their patchy distribution in the plasma-membrane of astrocytes is suggested to be involved in vivo in acute brain pathophysiologies (hypoxia/ischemia, edema). Currently available in vitro models hamper to study astroglial membrane channels in an in vivo-like expression pattern since their distribution is more mislocalized in the cytosol as well as in the whole plasma membrane. We sought to overcome this issue by using nanostructured materials, processed as film, named hydrotalcite nanoparticles (HTlc NP). Due to their chemical composition and ordered lamellar structure, HTlc enable hosting and controlled delivery of wide range of molecules (including peptides, siRNAs and oligonucleotides). Thus they have a huge potential as in vitro substrates to be used for interaction with neural cellular systems. Here, we evaluated the effect of the interaction of HTlc NP films on primary cultured rat neocortical astrocytes. Molecular and functional properties of astrocytes grown on HTlc films were compared to those of astrocytes grown on Poly-D-Lysine (PDL). Cell viability assay showed that HTlc-films supported astrocytes adhesion and survival with value comparable to those of PDL. Morfological analyses revealed that astrocytes grown on HTlc NP films displayed a starlike morphogy typical of differentiated astrocytes in vivo. Western blot (WB) analyses and immunofluorescence (IF) confocal imaging revealed that stellation was not accompanied by GFAP upregulation, indicating that differentiation was not due to occurrence of gliotic reaction. WB, IF and whole cell patch-clamp revealed that differentiation was accompanied by molecular and functional up-regulation and polarized membrane expression of both inward rectifying potassium channel Kir 4.1 and aquaporin 4, AQP4. Collectively, these results indicate that HTlc NP films are suitable substrates to generate novel in vitro models where to modulate, alter and study the specific functionality and expression of membrane channels of astrocytes. Sponsored by: Futuro in Ricerca (MIUR) RBFR12SJA8, EU-ITN-OLIMPIA GA 316832, Progetto Bandiera Fabbrica del Futuro Silk-it